The present invention is directed to small organometallic probes, processes for making the small organometallic probes, and applications of the small organometallic probes. In particular, the small organometallic probes of the present invention are preferably less than 0.02 micron (0.02 xcexcm) or 20 nanometers (20 nm) in diameter, and comprise a metal cluster compound having a solid metal core, with organic groups attached to the metal core so as to impart desirable physical and chemical properties to the organometallic probes. Alternatively, the organometallic probes may comprise a metal colloid having organic groups attached to the outer surface of the metal colloid. The metal clusters or colloids may also be functionalized with other molecules attached that can be used for targeting and detecting another substance, generally, a biologically significant substance, such as an antibody, a protein, or lipid bilayer. The metal in the metal clusters or colloids is gold, platinum, silver, palladium or combinations thereof.
In one specific embodiment, the metal in the cluster is palladium or platinum and the attached organic groups are covalently attached through 1,10-phenanthroline moieties. Also disclosed is a new method for making the small organometallic probes. The method consists of a chemical procedure wherein the metal cluster compounds are prepared directly by reaction of a mixture of a salt of the cluster metal and the coordinating organic groups with a reducing agent in solution.
Previous work by others has also described the preparation of gold and silver colloids. Such colloids do not have a fixed number of metal atoms and vary considerably in size. For example, the metal colloids can vary in size from 1 nm to 2 xcexcm in size and may contain from about 10 metal atoms to thousands of metal atoms, depending on size. It was found that a number of proteins, such as IgG antibodies, could be adsorbed to these sol particles.
Gold colloids have been most commonly described. These conjugates have been used in electron and light microscopy as well as on immunodot blots for detection of target molecules. These conjugates have many shortcomings. Since the molecules are only adsorbed onto the colloids, they also desorb to varying extents. This leads to free antibody which competes for antigen sites and lowers targeting of gold.
Furthermore, the shelf life of the conjugates is compromised by this problem. The xe2x80x98stickyxe2x80x99 colloids also tend to aggregate. If fluorescence is used to detect the target molecules, the gold particles quench most of it. Also, the gold colloids must be stabilized against dramatic aggregation or xe2x80x98flocculationxe2x80x99 when salts are added by adsorbing bully proteins, such as bovine serum albumin. Due to the effects of aggregation and bulky additives, the penetration of immunoprobes into tissues is generally  less than 0.5 xcexcm. Access of the probes to internal cell structures, e.g., nuclear proteins, or to cells deeper in a tissue sample, is impeded by these properties.
Colloidal gold immunoprobes are also used in diagnosis on immunoblots. The sensitivity of these detection schemes is also reduced by problems relating to detachment of antibodies from the gold which results in a short shelf life and non-specific gold binding causing problems with background signal. The gold prepared in standard ways also has low activity due to few adsorbed antibodies and denaturation of some antibodies during adsorption.
Various metal cluster containing organic shells have also been previously described, such as Au11 (PPh3)7Cl3 (PPh3=triphenylphosphine), and Pd561 L36O200 (where L=1,10-phenanthroline). These metal clusters have a fixed number of metal atoms in their metal cores which range in size from ca. 0.8-2.4 nm. Most of these metal clusters are based upon reduction of metal-triphenyl phosphine or the use of 1,10-phenanthroline.
Examples of larger cluster complexes (greater than 1 nm in size) have also been reported such as clusters having the formula M55(PPh3)12X6 (Ph=phenyl or m-phenylsulfonyl) where M=gold, platinum and rhodium and X=halide.
For example, Barlett, P. A. et al, in xe2x80x9cSynthesis of Water-Soluble Undecagold Cluster Compounds . . . ,xe2x80x9d J. Am. Chem. Soc., 100, 5085 (1978), describe a metal cluster compound (Au11) having a core of 11 gold atoms with a diameter of 0.8 nm. The metal core of 11 gold atoms in the undecagold metal cluster compound is surrounded by an organic shell of PAr3groups. This metal cluster compound has been used to form gold immunoprobes, for example, by conjugating A11 to Fabxe2x80x2 antibody fragments as well as other biological compounds.
Another metal cluster compound which has been used as a probe is Nanogold(trademark) available from the assignee of the present application. Nanogold(trademark) has a metal core with 50-70 gold atoms (the exact number not yet being known but believed to be 67 gold atoms) surrounded by a similar shell of organic groups (PAr3) such that Ar is an aryl group into which a reactive group such as a primary amine, a maleimide, or a N-hydroxysuccinimide ester may be incorporated for conjugation to biologically significant entities including antibody IgG molecules and Fabxe2x80x2 fragments, proteins, lipids, hormones and oligonucleotides. Nanogold(trademark) and the smaller undecagold cluster, which contains 11 gold atoms, have been used as probes for detecting and identifying biomolecules. The metal core of Nanogold(trademark) is 1.4 nm in diameter. The production of Nanogold is described in pending application Ser. No. 988,338, filed Dec. 9, 1992, of James F. Hainfeld and Frederic R. Furuya.
Another class of cluster complex compounds having Pt or Pd as the metal core and further having a core ranging in diameter from 1.8 to 3.6 nm is prepared by reduction of metal acetate in acetic acid by molecular hydrogen in the presence of 1,10-phenanthroline ligands. The ligated cluster is then carefully oxidized with air to neutralize the exposed metal atoms and render the compounds air-stable.
Complexes prepared by the above method and characterized by electron microscopy in order to determine the size of the metal core include a 1.81 nm core diameter platinum compound of proposed formula [Pt309phen36O30xc2x110] (see Scmidt, G., Morun, B., and Malm, J.-O.; Angew. Chem. Int. Ed. Eng., 1989, 28, 778), a 2.43 nm core diameter palladium compound of proposed formula [Pd561phen36O200] (de Aguiar, J. A. O.; Brom, H. B.; de Jongh, L. J., and Schmid, G.; Z. Phys. D.:Atoms, Molecules and Clusters, 1989, 12, 457), and a mixture of 3.16 and 3.6 nm core diameter palladium compounds with proposed formulae [Pd1415phen60Oxcx9c1100] and [Pd2057phen84Oxcx9c1600] respectively (Schmid, G.; Harms, M.; Malm, J.-O.; Bovin, J.-O; van Ruitenbeck, J.; Zandbergen, H. W., and Fu, W. T.; J. Amer. Chem. Soc., 1993, 115, 2046), where phen is either 1,10-phenanthroline or bathophenanthroline, (1). The proposed formulae are based upon the extension of the crystal packing of metal atoms within known smaller clusters outward in discrete layers.
Although the preparation and properties vary for these metal cluster compounds having organic shells, many of these can only be synthesized in low yields, derivatization for use in coupling to biomolecules is expensive in time and effort, and again in low yields, and many of the cluster compounds are degraded rapidly by heat or various chemical reagents.
In accordance with the present invention, a new class of metal cluster compounds, and a process for making such compounds, are described. The compounds may be generally described as organothiol metal clusters, wherein the metal core is comprised of gold, platinum, silver, palladium or combinations of these metals. Prominent among the disclosed organometallic compounds is a large palladium and platinum cluster compound. The metal core of the compounds, wherein Gold is the prominent metal, is about 1.4 nm in diameter and comprises about 50 to about 70 metal atoms. There are about 12 metal atoms on the surface of each cluster, and each surface metal atom is bound to an organic group by a thiol (Mxe2x80x94S) bond.
In another of its a the present invention is directed to mixed metal colloids and to a process for making such mixed metal colloids. While heretofore single metal colloids have been known (see description above), up to now no one has described a method for making a metal colloid with a combination of different metals the metals being selected from the group consisting of gold, silver, platinum, palladium and combinations thereof.
In another of its aspects, the present invention is directed to organic coated metal colloids, i.e., metal colloids surrounded by a shell of organic groups which are suitable for further functionaliation and covalent linking to other molecules. A process for producing such organic coated metal colloids is also described.
In another of its aspects, the present invention is directed to the organometallic clusters including the large palladium and platinum compounds described above which are covalently attached to various fluorescent molecules. This enables the preparation of dual-labeled organometallic probes which may be used to detect biomolecules using two distinct and different methods. A process for producing these organometallic probes is also described.
In another of its aspects, the present invention is directed to the organometallic clusters, including the large palladium and platinum compounds or colloids which are covalently attached to lipid molecules, and to a process for producing such compounds. The present invention is also directed to the use of such metal labeled lipids to form micelles and vesicles which are used in sensitive immunoassays, metal delivery in vivo, or other uses.
In another of its aspects, the present invention is directed to a stain based on the organometallic clusters or colloids described above, which stain may be used, for example, to stain proteins or nucleic acids after electrophoresis in gels.
In another of its aspects, the present invention is directed to the diagnostic and therapeutic medical uses of the metal conjugates described above. For example, the metals that are used can be radioactive, positron emitting, have unpaired electrons for magnetic resonance detection, or used with x-rays for absorptive or x-ray induced fluorescence detection. Other detection methods are also possible, such as mass spectroscopy. The organometallic particles may be attached to antitumor antibodies, or other targeting materials such as peptides, nucleic acids, or hormones, and used for sensitive diagnosis in vitro or in vivo. Since some isotopes produce radiations suitable for therapy or can otherwise be activated, e.g., by neutron activation, these may be used as therapeutic agents.
In another of its aspects, the present invention is directed to the development of the organometallic conjugates described above with silver developers or other toners and dyes for enhanced sensitivity or improved detection.
In yet a further aspect of the present invention, the organometallic probes described above may be used for superior ultrasensitive detection of substances, e.g., antigens or pollutants, when coupled with their use in, e.g., piezoelectric crystal mass measuring devices, detection based on changes in reflection from a surface where the particles bind, on blots (dot blots, Western or Southern blots), or by use of light, fluorescent, confocal, or electron microscopy.
The new class of organometallic cluster compounds described herein is synthesized by a novel approach and incorporates many metals such as gold, silver, platinum, and others as well as mixtures of these metals. Aside from being a novel class of clusters formed by a new process, these clusters are stable to 100xc2x0 C. or higher heating, in sharp contrast to the previously known triphenyl phosphine type clusters that are of a similar size which decompose at 55xc2x0 C. This heat stability creates many new areas of use for these compounds inaccessible with previous technology. Furthermore, the process is highly efficient and rapid in contrast to other synthetic routes currently known.
The field of lipids and liposomes is quite large, and covers basic biomedical research studies, diagnostics, cosmetics, drug delivery, food, catalysis and many other fields. The covalent attachment protocol described herein permits the correct attachment of the present organometallic clusters to lipids, and hence their incorporation into liposomes. They are stable, so that they may be used in a variety of further preparations of vesicles, micelles, or other constructs.
Also described herein for the first time are phospholipids and fatty acids covalently attached to gold particles. Furthermore, other lipids and lipid-like compounds can be used as well as other organometallic particles, such as the others described herein. Previously, it has not been possible to prepare gold particles conjugated to lipids. By using a covalent attachment, the products formed are indefinitely stable and can be handled in a variety of conditions for further synthesis of vesicles, micelles, or other constructs.
Also described herein is an extremely sensitive lipoimmunoassay (LIA) based on these organometallic liposomes. This improves the sensitivity and stability of currently available lipoimmunoassays.
One current form of the LIA is to encapsulate fluorescent molecules within a liposome that contains an antigen in its lipid layer. The concentration of fluorescent molecules is high enough within the vesicles such that their fluorescence is quenched. When these vesicles are exposed to serum containing the specific antibody to the antigen on the liposome and complement, the vesicles rupture and release the fluorescent molecules. When they disperse in the medium they are diluted and no longer self quench and the increase in fluorescence is measured. This test has several practical flaws. Leakage of the fluorescence over time is observed, and sensitivity could be improved. The metal-lipid conjugates disclosed in this application overcome both of these shortcomings. First, gold or another metal is formed into the unique organometallic clusters or colloids that are disclosed in this application. They are then reacted to form covalent bonds with lipid molecules. These are then treated in the usual way to form liposomes (i.e., by sonication, ether bubbling, wall peeling above the lipid phase transition temperature, etc.). This will form vesicles with, e.g., gold particles both on the inner and outer surfaces of the liposomes. The gold on the outer surface is selectively removed by treatment with potassium cyanide, xcex2-mercaptoethanol, or other effective treatments that disintegrate the gold particles. The internal gold particles are not affected due to the lipid barrier to these reagents. After centrifugation or other purification, the vesicles (also containing antigen) are mixed with test serum as described above containing silver developer. When the vesicles lyse, the internal gold particles are exposed to the developer and a strong black color is produced. Since the gold is covalently attached, the leakage problem is circumvented and storage is greatly improved. Since silver development of gold particles has been shown to be more sensitive than fluorescence, the sensitivity is improved.
Polyacrylamide gel electrophoresis for proteins, nucleic acids and other substances is widely used in research and diagnostics. Variations are use of other gels, such as agarose, transfer of products onto immobilizing membranes, and use of probes such as nucleic acids (Southern) or antibodies (Western) to identify specific bands. In general, the bands at the end of the run are invisible and must be stained in some fashion. The two most popular protein stains are Coomassie Blue and a silver stain. Because of the weak binding to the target material over the gel, lengthy times of exposure and washing are needed, taking from 1 to 16 hours. Also, a number of steps are involved. The new stain we describe herein is vastly improved in two ways: a) development time is reduced to about 1-5 minutes, and b) the sensitivity is far greater than the other stains available. This advancement is achieved using novel organometallic compounds that more strongly interact with the target material, followed by the improved development which nucleates specifically on these organometal particles.
The use of nucleic acids (DNA and RNA) in research and medicine is very important. Many diagnostic tests are now based upon recognizing specific genetic sequences, and cloning, PCR and other molecular genetic techniques have contributed to the widespread and multifaceted applications in this area. Probes used for such tests utilize chemically attached haptens, labels or other entities which generate a signal, or are bound by other signal-producing probes. More sensitive probes are needed in order to de specific conditions earlier, using less biopsied material. The covalent attachment methods described herein are also applicable to the preparation of nucleic acid probes with the same advantages described for the antibody conjugates, and metal-containing nucleic acids may be used for other purposes such as genetic material purification.
Described herein are methods to incorporate organometallic particles into nucleic acids to provide extremely sensitive assays based upon hybridization. These metal containing nucleic acids may also be used for many other purposes, such as genetic material purification.
Labeled targeted biomolecules form the basis of diagnostic tests for many diseases and conditions. The probes of the present invention have demonstrated sensitivities significantly greater than many currently used technologies, including radioactive labeling, fluorescent labeling and colloidal gold. The increased sensitivity of the present claimed probes, most notably, those probes of the embodiment wherein the metal in the cluster is palladium or platinum, will, in turn, allow for earlier detection of harmful infections or conditions, with fewer antigen test strips needed, fewer false-positive results, and smaller biopsied specimens. Furthermore, the present invention avoids the use of radioactive or highly toxic materials, which are very costly and difficult to dispose of and impose limitations on many currently used technologies.
Unique improvements in a number of areas are now possible with the new organometallic probes described herein. For example, in medicine, molecular probes are used for diagnostic and therapeutic applications. The superior qualities of the conjugates described herein such as, covalent coupling, improved higher specific activity, various modes of detection (fluorescence, silver development, electron microscopy, X-rays, etc.) and improved sensitivity, should make these excellent candidates to replace many diagnostic detection schemes. Radioactive metals used in these clusters, colloids, and conjugates can be used for improved delivery of diagnostic or therapeutic radiation, e.g., by using antitumor antibodies. Use of positrons and other modes of detection are enhanced by the improved performance of these unique conjugates.
More specifically, current tests based on immunology are only able to detect a pathological condition after a certain concentration of antigen is present. For most conditions, such as AIDS, diagnosis at an earlier stage than current tests are capable of is important. Also, some patients have lower antibody titers and are more difficult to detect. The higher specific activity of novel conjugates described herein and their higher sensitivity in comparable tests with existing methods mean that they overcome an important shortcoming of the current technology. A further consideration in diagnosis and medicine (and most other applications) is the cost and speed of the tests performed (overall materials and labor). Since the conjugates described herein are more sensitive, fewer antigens on test strips need to be used, and fewer reagents need be used. They also develop faster than current tests due to their high sensitivity, thus taking less time to use. Since sensitivity is greater than comparable radioactive probes, more biohazards and pollutants could be eliminated by use of the conjugates described herein.
In vivo diagnostics currently also have shortcoming of sensitivity, cost, toxicity, biohazard, and environmental waste generation. As just one example, radioisotopes attached to drugs or antibodies are used, which subject the patient to radiation. The conjugates described herein can, for example, be used non-radioactively and imaged using x-ray absorption or x-ray induced fluorescence and computer tomography, giving higher resolution and lower dose to the patient.
For therapeutic applications, current technologies suffer from limitations. As just one example, radioimmunotherapy of cancer has not been thoroughly successful for a number of reasons. Enough of a suitable radioisotope must be selectively delivered to the tumor cells. Gold-189/199 is an excellent choice because of its intermediate xcex2 emission and 3 day half life. Unfortunately, it has not been stably conjugated to antibodies since it does not chelate to the usual metal chelators such as DTPA. Some progress has been made using undecagold clusters but these have been shown to have high uptake by the kidneys and show some degradation in the serum with time. One of the processes and products described in this application is a colloidal gold to which many Fabxe2x80x2 antibody fragments can be covalently attached. This has a number of advantages for this application in radioimmunotherapy: a) this gold exhibits excellent stability properties, b) it has multiple Fabxe2x80x2 fragments attached which improves immunoreactivity of the conjugate yielding better targeting, c) the multiple Fab""s per gold particle provide a redundancy in design so that if one or more antibody fragment loses its activity either by denaturation, radiation, gold binding, or other factors, the remaining intact antibody fragments can still serve to target the radioactive gold to the tumor; d) the gold particle consists of xcx9c100,000 gold atoms, and this number is design dependent and can be varied. The large number of isotopes carried to the tumor per antibody binding site is huge compared with other proposed radioimmunotherapies that use only one isotope per antibody. This means that orders of magnitude more dose or specific activity per antibody can be delivered. This is an important factor in achieving successful therapy. A number of significant advantages are therefore possible in this area by the conjugates disclosed herein. Other therapies, such as arthritis treatment using gold, would also be improved by the unique design, flexibility, and advantages of the novel gold structures herein disclosed.
A number of detection schemes have been devised that have improved the sensitivity or economics. One such advance is the piezoelectric detector. One mode of operation is to coat a piezoelectric crystal surface with an antigen. The crystal is part of an oscillator and its frequency of oscillation is affected by the crystal mass. When an antibody (from, e.g., test serum) binds to this surface layer, the additional molecules change the mass slightly which can be detected via a frequency change. By using, e.g., the gold conjugates described herein, e.g., the colloidal particles with covalent antibodies attached, a further solution containing gold-anti-human antibodies could be attached to the primary antibodies bound to the surface (if present in the serum) to form a xe2x80x9csandwichxe2x80x9d. The large mass of the gold (xcx9c5xc3x97107 compared with 1.5xc3x97105 for IgG) will greatly amplify the signal making detection levels far lower than with existing methods. A related technique uses reflection of light from a surface. When the surface is coated with a layer, even of antibody molecules, there is a change in the peak reflection angle. Use of the metallic particles (as just described) will influence to a far greater extent the change in reflection due to the strong optical properties of gold or other metal particles used. Choice of wavelength, polarization and optimizing other parameters for metal particle interaction and detection can further enhance the sensitivity.
The greater reactivity of the organometallic covalent probes can also be used to improve the detection sensitivity in other known schemes or instruments. Use in blot tests with silver enhancement of metal particles improves sensitivity over current technologies. Use of light and confocal microscopes as well as scanning and transmission electron microscopes will also benefit from these new probes which have advantages in sensitivity, small size, and high specific activity.
In accordance with one specific embodiment, a new form of large palladium and platinum compounds in which the coordinated organic groups have been modified to impart water-solubility and to enable covalent chemical conjugation to biologically important entities. The metal core ranges from about 1.8 to 3.6 nm in diameter and comprises from about 309 to out 2057 atoms. From about 36 to about 84 organic moieties, specifically 1,10-phenanthrolines, are bound to the surface metal atoms through Mxe2x80x94N bonds, and in addition the palladium or platinum atoms are believed to be bound to between approximately 30 and 1,600 oxygen atoms. Platinum compounds larger than 1.8 nm have been observed by electron microscopy which have not been previously described. The diameter of the metal cluster, inclusive of the metal core and the organic moities ranges from about 3 to about 5 nm.
The present invention is also directed to a new method for preparing the large palladium and platinum organometallic cluster complexes described above. The new process utilizes a simpler and less hazardous procedure than currently disclosed methods. It also utilizes milder preparative conditions, which permit the introduction of a variety of modified organic groups which impart novel properties and reactivity to the clusters.
The new larger palladium and platinum organometallic compounds described above are synthesized using organic groups which confer water-solubility to the complexes and enable covalent cross-linking of the complexes to other reactive molecules, without introducing ionic charges. This feature has heretofore never been described in prior art literature. A distinct advantage of this feature is that it creates numerous opportunities for application of the above noted cluster complexes to areas which were previously inaccessible with conventional probes.
Also, the preparative process for preparing the above noted large palladium and platinum compounds results in the efficient and rapid synthesis of the contemplated compounds. While prior art processes for making conventional gold probes include dissolving reagents in acetic acid, the process of preparing the large palladium and platinum compounds of the present invention comprises dissolving the reagents in inert organic solvents, which, in turn, allows for the use of many different organic groups, which were previously incompatible with conventional methods. An added advantage realized by the large palladium and platinum compounds is that it eliminates potential hazards associated with the use of acetic acid.
A further contrast between the process noted above and prior art methods is that while previous procedures utilize hydrogen gas as the reagent to form clusters, the process of the preparing the large palladium and platinum compounds of the present invention comprises dissolving reducing agents such as sodium borohydride. This feature, simplifies significantly, the overall experimental complexity while reducing the overall expense and hazards of handling of compressed and/or flammable gas (hydrogen), which are the hallmarks of conventional processes.
The uncharged nature of the large palladium and platinum organometallic cluster probes, together with their ability to covalently link to a targeting agent also circumvents some of the same difficulties associated with colloidal gold probes of similar size.
Prior combined fluorescent and metal particle probes, e.g., where a gold particle has a fluorescent molecule attached to it, which is then conjugated to, e.g., an antibody, have been notoriously unsuccessful. This drawback can be traced to the strong quenching of fluorescence by the colloidal gold (which absorbs strongly in the visible region), together with the difficulty associated with preparing such conventional probes.
Methods for producing conventional metal particle probes have generally been fraught with the same difficulties that have visited colloidal gold probes such as their inherent tendency to xe2x80x9cstickxe2x80x9d or cause molecules of interest to adsorb to their respective surface, thereby preventing the molecule of interest to remain bound to the probe.
Generally, colloidal gold particles, instead of being bound covalently to the target molecule, usually adsorb electrostatically to the targeting agent. As such, these gold particles readily disassociate from the targeting agent, which, in turn, results in low labeling. Conventional colloidal gold particles are also known to xe2x80x98stickxe2x80x99 together and form large aggregates, which reduces their access to the target agent. These same gold particles have been known to adhere to components of the system they are used to investigate which, in turn, results in non-specific binding.
Described herein is a novel method of covalently linking fluorescent molecules to small organometallic particles which include the large palladium and platinum compound clusters described above. This circumvents the difficulties of the previous technology in two significant ways: first, the fluorescent molecule and the antibody or other targeting molecule (if desired) are covalently attached and do not readily xe2x80x9cdesorb.xe2x80x9d This attachment can be performed in mild physiological buffers, thus eliminating the very low ionic strength conditions necessary for colloidal gold conjugation. Thus, molecules difficult to attach to colloidal gold are simply and more stably attached by this covalent route.
Second, the chosen metal particle does not significantly quench the fluorescence, in sharp contrast to colloidal gold. In many cases, full fluorescent activity is maintained. The success of these new dual conjugates (combining fluorescence and metal, e.g., gold) permits unique applications such as fluorescent immunolabeling which is discernible by light or confocal microscopy; when cells exhibiting optimal distribution of the probe are identified, these may be processed for electron microscopy so that high resolution ultrastructure localization of antigens may be-performed. By using a dual label, there is no question as to the distributions being identical. This type of probe has long been sought by cell biologists.
Combined metal cluster and fluorescent probes have long been sought by cell biologists. As noted previously, a known disadvantage of conventional colloidal gold probes is that these probes exhibit significant fluorescence quenching and profound dissociation. This drawback can be traced to the smallness in size of conventional gold probes. As a result, it is often difficult to view these clusters in some electron microscopy applications, particularly where other electron-dense stains are used. An added advantage of the present invention, particularly the organometallic probes containing palladium or platinum is that they can be readily observed in the electron microscope even where other such stains are used. This feature is not inherent in conventional metal probes.
The highly sensitive probes of the present invention are also good candidates for use as reagents in research. The small probes make it easier to identify specific antigens in biological specimens. In addition, the small size of the organic groups of the large palladium and platinum organometallic compounds reduces the overall size of the probe, compared with prior art colloidal gold probes. This feature, in turn, allows for easy penetration of cells and tissue sections.
An added advantage of using the above noted probes in the medical and diagnostic fields is that the small size of the probes provides them with easy access to hindered sites, which is not possible with conventional probes.
For example, when comparing the probes of the present invention with Nanogold(trademark), which is currently available from the assignee of the present application, it was noticed that Nanogold(trademark) was able to penetrate up to 30 microns (30xcexc) into tissue sections, while the gold colloid probes of nominally similar diameter penetrated only 0.5 microns (0.5xcexc) of the tissue section. Enhanced penetration into cell nuclei was also observed with the probes of the large palladium and platinum clusters, while a comparably sized conventional colloidal gold probe did not access the cell nucleus at all.
A brief description of the various terms appearing in the text of the present application appears hereinafter.
1. xe2x80x9cLarge Palladium and Platinum Clustersxe2x80x9d are cluster complexes of palladium or platinum in which the central metal core is between about 1.8 nm and about 3.6 nm in diameter and contains between approximately 309 and approximately 2,057 metal atoms. The surface atoms are bound to a variety of substituted 1,10-phenanthroline ligands, numbering from about 36 to about 84 in total, and also to oxygen atoms, believed to number between about 30 and about 1,600, which stabilize the surface and block further surface reactions. The diameter of the metal cluster inclusive of the internal metal core and the organic moities varies from about 2 to about 5 nm.
2. The 1,10-phenanthroline ligands used to prepare the large palladium and platinum clusters, listed above in #1, are modified by the introduction or specific chemical functional groups at either the 4 and 7-positions, or the 5-position. These substituents are capped either with reactive groups, including primary amines, maleimides, and sulfo-NHS esters, which can be covalently bound to other molecules, or with solubilizing groups including 1,2-diols or p-phenylsulfonates, or with biologically compatible but inert functionaries including N-methylcarboxamides or acetamides.
3. Reactive groups incorporated into coordinated ligands may be used to attach the cluster covalently to antibodies, proteins, lipids, peptides, drugs, DNA, RNA, or any other biologically active molecule with a group which may be cross-linked. This circumvents the requirements for low salt concentrations which prevent conjugation of many molecules-such as IgMs-with colloidal gold.
4. Combined (bifunctional) fluorescent and metal particle probes comprising the large palladium and platinum cluster listed above (according to #1) can be synthesized having the formula Mn(OrF)m(Orxe2x80x2T)l(Orxe2x80x3)pOq, where M is the metal core consisting of platinum or palladium of which the surface atoms may be covalently bonded to a shell of organic groups (Or, Orxe2x80x2, Orxe2x80x3). Or and Orxe2x80x2 are covalent coupling moieties, specifically 1,10-phenanthrolines containing cross-linkable groups such as amines, carboxyls, maleimides or N-hydroxysuccinimide esters, F is a fluorescent molecule (fluorescein, Texas Red, rhodamine, aminomethyl coumarin, etc.), and T is an optional targeting molecule such as an antibody, peptide, drug, etc. Or, Orxe2x80x2 and Orxe2x80x3 may be the same or different. The subscripts l, m, n. p and q indicate that multiple copies of each moiety are possible and include mixtures of different fluorescent groups, organic groups and targeting molecules for multifunctional conjugates. A specific example is the following:
[Pt309(2)18(3)9(C12H6N2xe2x80x94COxe2x80x94NHxe2x80x94CH2CHCH2NHxe2x80x94F)9O30]
wherein C12H6N2xe2x80x94COxe2x80x94NHxe2x80x94CH2CH2CH2NHxe2x80x94F refers to a ligand exemplified by formula 3, after reaction of the primary amine with an activated carboxylic ester of fluorescein.
5. A new method for the chemical preparation of cluster complexes according to #1 comprises dissolving a divalent salt of palladium or platinum MXXxe2x80x2, wherein M is palladium or platinum and X and Xxe2x80x2 are acetate, chloride, bromide, iodide and acetyl-acetonate anions, and a mixture of Or, Orxe2x80x2and Orxe2x80x3 in the mole ratio desired in the final cluster, in either N, Nxe2x80x2-dimethylacetamide or in a mixture of ethanol, benzene, dichloromethane and water. Thereafter, the resulting product is reacted with a solution of a reducing agent such as sodium borohydride or a hydride-transfer reagent of general formula Mxe2x80x2EHRRxe2x80x2Rxe2x80x3 where Mxe2x80x2 represents sodium or lithium, E represents boron or aluminum, H represents hydrogen, and R, Rxe2x80x2 and Rxe2x80x3 are the same or different and are hydrogen or straight-chain or branched,cain hydrocarbons with 1-10 carbon atoms, dissolved in N, Nxe2x80x2-dimethylacetamide or in another inert solvent.
The above process, essentially, eliminates the hazards and experimental complexity associated with using hydrogen gas or acetic acid, both of which are required by the conventional technology. Also, the reaction conditions are compatible with a variety of Or, Orxe2x80x2 and Orxe2x80x3 groups or their-precursors.
6. After cluster formation, cross-linking to other molecules T may be achieved by a variety of methods. If Orxe2x80x2 is a primary amine-containing moiety, this is converted to a maleimide or N-hydroxysuccinimide ester and reacted with a molecule which contains either a thiol or a primary amine; alternatively, it may be reacted directly, for example with an activated ester or with an aldehyde group in the target molecule. This results in the formation of a targeted probe which may either be visualized directly in the electron microscope, or rendered visible in the light microscope or other optical systems by a process of silver enhancement.
7. xe2x80x9cThiol gold clustersxe2x80x9d are novel gold dusters produced by a novel synthesis. The procedure is: form an organic-gold complex by reacting a compound containing a thiol with gold in solution. A second equivalent is also added of the thiol compound. Finally the gold organic is reduced with NaBH4 or other reducing agents and organometallic particles are formed. These have the general formula AunRmRxe2x80x2l . . . , where n, m, and l are integers, R and Rxe2x80x2 are organic thiols, (e.g., alkyl thiols, aryl thiols, proteins containing thiol, peptides or nucleic acids with thiol, glutathione, cysteine, thioglucose, thiolbenzoic acid, etc.) and the ellipsis indicates that one or more organic thiols may be used. With two equivalents of organic thiol compound, clusters with gold cores xcx9c1.4 nm are formed with many organic moieties. The organic moiety may then be reacted by usual reactions to covalently link this particle to antibodies, lipids, carbohydrates, nucleic acids, or other molecules to form probes. Mixtures of organic thiols may be used to provide mixed functionality to the clusters. These organo-gold clusters are stable to heating at 100xc2x0 C.
8. Combined (bifunctional) fluorescent and metal particle probes wherein the metal in the metal clusters is gold, platinum, palladium, silver or combinations thereof (according to #7, described above) have been synthesized and have the following formula:
Mn(OrF)m(Orxe2x80x2T)l(Orxe2x80x3)p
wherein M is a metal core consisting of multiple metal atoms (Au, Pt, Ag, Pd) that can be mixed, covalently bonded to a shell of organic groups (Or, Orxe2x80x2, and Orxe2x80x3). Or and Orxe2x80x2 are organic coupling moieties, (e.g., triphenyl phosphine 1,10 phenanthrolines, triphenyl phosphine or phenanthrolines containing linkable groups such as amines or carboxyls, triphenyl phosphine or 1,10, phenanthrolines containing reactive groups such as maleimides or N-hydroxysuccinimide esters, P(C6H4xe2x80x94COxe2x80x94NHxe2x80x94(CH2)3xe2x80x94NH2)3, P(C6H4xe2x80x94COxe2x80x94NHxe2x80x94CH3)2(CH6H4xe2x80x94COxe2x80x94NHxe2x80x94(CH2)3xe2x80x94NH2), P(C6H4xe2x80x94COxe2x80x94NHxe2x80x94(CH2)3xe2x80x94NC4O2H2)3, P(C6H4xe2x80x94COxe2x80x94NHxe2x80x94(CH2)3xe2x80x94NHxe2x80x94(CH2)6xe2x80x94CO2xe2x80x94NC4O2H4)3, etc.), Orxe2x80x3 is an organic group (e.g., triphenyl phosphine, 1,10 phenanthrolines, P(C6H4xe2x80x94CHOHxe2x80x94CH2OH)3, P(C6H4xe2x80x94COxe2x80x94NHxe2x80x94CH3)3, P(C6H5)3, P(C6H4SO3)3, etc.), part of the metal cluster, F is a fluorescent molecule, (e.g., fluorescein, rhodamine, aminomethyl coumarin, Texas Red, etc.) and T is an optional targeting molecule such as antibody, peptide, drug, etc. Or, Orxe2x80x2, and Orxe2x80x3 may be the same or different. The subscripts l, n, m and p indicate that multiple copies of each moiety are possible and include mixtures of different metals, fluorescent groups, organic groups and targeting molecules for multifunctional conjugates. A specific example of this is:
Au11[Pph(CONHCH3)]6phCONH(CH2)3NHxe2x80x94F
wherein ph is a phenyl group, and F is a fluorescent molecule.
A process to produce the above noted multifunctional metal particle according to #5, supra, comprises synthesizing a metal cluster or metal colloid having one or more reactive groups such as an amine or carboxyl. The particles are thereafter reacted to covalently link a fluorescent molecule such as fluorescein, Texas Red, rhodamine, or aminomethyl coumarin, and optionally a targeting or other molecule is covalently attached such as an antibody or antibody fragment, a peptide, streptavidin, or other proteins, a nucleic acid (RNA or DNA), drugs, hormones, or other molecules. Alternatively, the fluorescent groups may be incorporated into ligands which are then used to prepare the cluster.
9. The thiol-gold preparation described in #8 above may be altered such that a larger molar ratio of organic thiol to gold is used. Ratios above approximately 2:1 or below 1:2 result in organic-gold colloids whose size depends on this ratio. These are useful when large gold particles are desired.
10. The organic thiol-gold preparations described in #""s 8 and 9 above may be made using a similar process with alternatives metals to gold, e.g., platinum, silver, palladium and other metals.
11. The organic thiol-metal particles described in #""s 8, 9 and 10 above may be made using mixtures of metal ions, e.g., gold and silver, resulting in mixed metal clusters.
12. A novel process has been developed for coating colloidal particles (of various types including gold, silver, palladium, platinum and other metals) with organic moieties having groups suitable for covalently attaching additional molecules, such as antibodies, nucleic acids, lipids, peptides, and other proteins. The process consists of synthesizing the metal colloid in the presence of a suitable polymer, e.g., HAuCl4 (0.01%) in 0.05 M sodium hydrogen maleate buffer (pH 6.0), with 0.004% tannic acid. The polymer may be chosen from a linear or branched group with functional groups attached, such as polyamino acids, polyethylene derivatives, other polymers, or mixtures thereof. Optimal molecular weight of the polymer varies with the specific ones chosen. A second method is to synthesize the metal particle first, e.g., by combining 0.01% HAuCl4 with 1% sodium citrate with heating. Once gold colloid is formed of the desired size, it is coated with one of the above polymers by mixing the two together and optionally warming to 60-100xc2x0 C. for several minutes. The polymer coating may be further stabilized by a) microwave heating, b) further chemical crosslinking, e.g., by glutaraldehyde or other linkers, or by continued polymerization adding substrate molecules for a brief period. Use of N, Nxe2x80x2-methylene bis acrylamide, for example, can covalently further stabilize the polymer coating. Photocrosslinking may also be used.
The functionalized polymer coating may now be used to covalently attach proteins, peptides, antibodies, lipids, carbohydrates, nucleic acids, drugs, hormones, or other substances. This has the advantage that this step may be done mildly, in physiological buffers if desired, using standard crosslinking technology. This eliminates the usual restriction that conjugation must be performed in very low ionic strength buffers, which precludes attachment of certain molecules such as many IgM""s which cannot withstand the low ionic strength requirement.
13. Lipid molecules (fatty acids, phospholipids, or others) are covalently attached to metal particles (clusters or coated reactive colloids). This process uses a reactive lipid derivative, e.g., a sulfonyl chloride or anhydride, which is reacted with an amino group, for example, on the metal particle. Alternatively, organic groups on the metal particle or lipid may be reacted with bifunctional crosslinkers which are then reacted with the other species. Another process is to pre-synthesize the organic components of the metal particles (e.g., phosphine or polymers) with lipid molecules attached and then to use these organo-lipids in constructing the functionalized metal particle.
The general formula for the product is:
Mxe2x80x94Orxe2x80x94L
wherein M is the metal particle (either cluster or colloid of Au, Pt, Ag, Pd, and combinations), Or is an organic group of the organometallic particle (such as, phosphine containing linkable groups, polymers containing linkable groups, P(C6H4xe2x80x94COxe2x80x94NHxe2x80x94(CH2)3xe2x80x94NH2)3, polyethyleneimine, polyacrylamide hydrazide, polylysine, etc.), and L is the lipid moiety.
14. A novel gel stain product is a metal (preferably gold) cluster of the form described in #8 above using appropriate organic thiols. The thiols are preferably o-thiol benzoic acid, glutathione, and thioglucose, although others may be used. The general formula describing the product is:
Mk(SOr)m(Sorxe2x80x2)n
where M is the metal, S is sulfur, Or and Orxe2x80x2 are organic groups (such as proteins or nucleic acids containing thiols, most other organic thiols, glucose, benzoic acid, glutathione, cholesterol, etc.), and k, m and n are integers. The ellipsis indicates that one or more different (Sor) groups (organic thiols) may be attached per metal core.
The process for a protein polyacrylamide gel is as follows: mix the organometallic particle with the protein sample with or without SDS (sodium dodecyl sulfate), for native or denaturing gels; if a reducing gel is desired, the protein is first reduced with a reductant, preferably xcex2-mercaptoethanol, followed by a thiol blocking agent, preferably iodoacetamide, before adding the metal stain. The sample is heated briefly (e.g., one minute), loaded onto the gel and run normally. Stain development is effected by soaking the gel in a silver enhancement medium (e.g., AgNO3 with hydroquinone) for several minutes followed by water or fixing washes.
15. The organometallic particles described above may be covalently incorporated into nucleic acids by several techniques. One is via synthesis of a metal particle attached to a nucleic acid base or analogue that has its other functional groups protected so that it is compatible with automated nucleic acids synthesis, such as use of phosphoramadite chemistry. A second approach is to incorporate appropriate organometallic base analogs into nucleic acids enzymatically. A third method is to react activated organometallic clusters or colloids with functional groups incorporated into nucleic acids (such as primary amines). A fourth approach is to use organometallic particles that contain photoactive group(s) that then covalently attach to the nucleic acids when light activated.
The present invention shall be described in greater detail with reference to the examples appearing hereinafter.